The thermodynamics of ligand binding to the GroEL chaperonin protein from E. coli is probed using isothermal titration calorimetry. GroEL is a heat-shock protein that assists other proteins to fold correctly both in vivo and in vitro . Under conditions where an unfolded substrate protein cannot spontaneously fold GroEL requires K+, Mg2+, ATP hydrolysis, and a co-chaperonin protein, GroES. The thermodynamics of each ligand binding will be examined individually and in combination with other ligands. From these studies, we hope to discover the thermodynamic effect of each ligand on the thermodynamics of unfolded proteins, and compare these results to the activation energy for protein folding. These studies will address one of the major unresolved issues of chaperonin protein folding mechanism: How does the chaperonin transfer the energy of unfolded protein binding and a nucleotide-induced conformational change to enhance the folding yield?